The original application for competitive renewal of the R01 grant that was selected for a MERIT award proposed three alms to address three fundamental questions relevant to the mechanisms by which CFTR forms Its functional, native structure and how this process Is altered by disease-causing mutations. How does ?F508 interfere with NBD1 folding? Does ?F508 significantly modify the interaction of the folded NBD1 with other domains? What interactions with quality control proteins are critical? During the first four and a half years of MERIT support we have answered the first two questions In an exploitable way. These results indicate that CFTR folding is a hierarchical process and provide a clear explanation for the efficacy ceiling for extant compounds that correct folding of the ?F508 mutant. They also suggest a means to a mechanism-based approach for the discovery new compounds that work in synergy with extant correctors or novel compounds that circumvent the ceiling. These approaches are already being employed by a number of drug discovery efforts. Finally, using a powerful specific photo-crosslinking method, differential interactions of proteins with mutant and wild type nascent chains translated in vitro have revealed a previously unappreciated mechanism for preemptive quality control. The system involves proteins that lead to the degradation of the mRNA coding for the mutant protein, thereby reducing the production of protein bound to misfold. This mechanism prevents the accumulation of potentially cytotoxic misfoided proteins without spending energy for futile translation and subsequent ATP dependent proteolysis by the proteasome. We are now requesting continued support of the MERIT award to extend the analyses successfully applied to ?F508 in Aim 1 and 2 to additional CF-causing mutations and to define and characterize the mechanisms responsible for preemptive quality control system discovered during execution of Aim 3. We thank the institute for selecting our study for MERIT support that allowed pursuit of the long term discovery effort of Aim 3 that has now revealed novel and unexpected biology. Such a path would not have been feasible under the time constraints of a R01.